This invention relates to the field of electroporation and mass transfer across cell membranes in general and the transport of ions across a cell membrane in particular.
Electroporation is a technique that is used for introducing chemical species into biological cells, and is performed by exposing the cells to an electric potential that traverses the cell membrane. While its mechanism is not fully understood, electroporation is believed to involve the breakdown of the cell membrane lipid bilayer leading to the formation of transient or permanent pores in the membrane that permit the chemical species to enter the cell by diffusion. The electric potential is typically applied in pulses, and whether the pore formation is reversible or irreversible depends on such parameters as the amplitude, length, shape and repetition rate of the pulses, in addition to the type and development stage of the cell. As a method of introducing chemical species into cells, electroporation offers numerous advantages: it is simple to use; it can be used to treat whole populations of cells simultaneously; it can be used to introduce essentially any macromolecule into a cell; it can be used with a wide variety of primary or established cell lines and is particularly effective with certain cell lines; and it can be used on both prokaryotic and eukaryotic cells without major modifications or adaptations to cell type and origin. Electroporation is currently used on cells in suspension or in culture, as well as cells in tissues and organs.
Electroporation is currently performed by placing one or more cells, in suspension or in tissue, between two electrodes connected to a generator that emits pulses of a high-voltage electric field. The pore formation, or permealization, of the membrane occurs at the cell poles, which are the sites on the cell membranes that directly face the electrodes and thus the sites at which the transmembrane potential is highest. Unfortunately, the degree of permealization occurring in electroporation varies with the cell type and also varies among cells in a given population. Furthermore, since the procedure is performed in large populations of cells whose properties vary among the individual cells in the population, the electroporation conditions can only be selected to address the average qualities of the cell population; the procedure as currently practiced cannot be adapted to the specific characteristics of individual cells. Of particular concern is that under certain conditions, the electrical potential is too low for a cell membrane to become permeabilized, while under other conditions electroporation can induce irreversible pore formation and cell death. A high electric field, for example, may thus produce an increase in transfection efficiency in one portion of a cell population while causing cell death in another. A further problem with known methods of electroporation is that the efficiency of transfection by electroporation can at times be low. In the case of DNA, for example, a large amount of DNA is needed in the surrounding medium to achieve effective transformation of the cell.
Many of the problems identified above are a consequence of the fact that the process of electroporation in both individual cells and tissues cannot be controlled in real time. There are no means at present to ascertain in real time when a cell enters a state of electroporation. As a result, the outcome of an electroporation protocol can only be determined through the eventual consequences of the mass transfer process and its effect on the cell. These occur long after the mass transfer under electroporation has taken place. These and other deficiencies of current methods of electroporation are addressed by the present invention.
Also relevant to the present invention are current techniques for the study and control of mass transfer across cell membranes. Knowledge of mass transfer across cell membranes in nature, both in cells that are functioning normally and in diseased cells, is valuable in the study of certain diseases. In addition, the ability to modify and control mass transfer across cell membranes is a useful tool in conducting research and therapy in modern biotechnology and medicine. The introduction or removal of chemical species such as DNA or proteins from the cell to control the function, physiology, or behavior of the cell provides valuable information regarding both normal and abnormal physiological processes of the cell.
The most common method of effecting and studying mass transfer across a cell membrane is to place the cell in contact with a solution that contains the compound that is to be transported across the membrane, either with or without electroporation. This bulk transfer method does not permit precise control or measurement of the mass transfer across the membrane. The composition of the solution at specific sites is not known and is variable. In addition, when an electric field is present, the local field intensity will vary from one point to another. Furthermore, the surface of the cell that is exposed to the solution is not well defined. Cell surface areas vary among cells in a given population, and this leads to significant differences among the cells in the amount of mass transfer. For these reasons, the amount of mass transfer achieved by bulk transfer processes is not uniform among cells, and the actual amount transferred for any particular cell cannot be determined.
Attempts made so far to overcome the limitations of bulk transfer techniques include techniques for treating individual cells that include either the mechanical injection (microinjection) of chemical compounds through the cell membrane or electroporation with microelectrodes. In injection techniques, the membrane is penetrated with a needle to deliver a chemical agent, localizing the application of the chemical agent to a small region close to the point of injection. This requires manipulation of the cell with the human hand, a technique that is difficult to perform, labor-intensive, and not readily reproducible. Electroporation with microelectrodes suffers these problems as well as the lack of any means to detect the onset of electroporation in an individual cell. These problems are likewise addressed by the present invention.
Devices, systems and particular methods are disclosed which make it possible to precisely monitor the movement of materials across a cell membrane. The information gained from monitoring the movement of materials across a cell membrane may be directly applied to deduce information with respect to the cell and/or its membrane. Alternatively, the information obtained from monitoring may be applied in order to control the movement of materials across the cell membrane such as by controlling the application of electrical current. Devices and systems of the invention make it possible to move charged molecules, and in particular ionic species, across a cell membrane and precisely monitor the occurrence of such. When carrying out electroporation using the devices, systems and methods of the invention the information obtained from monitoring the movement of the charged particles across the cell membrane is used to control the process of mass transfer across a cell membrane. Specifically, the system is used to obtain measurements and changes in electrical impedance across a cell membrane while the mass transfer properties of the cell are changed by the application of electrical current. Thus, information obtained on electrical impedance changes brought by the application of electrical current are used, in real time, in order to control the movement of charged molecules across a cell membrane.
One aspect of the invention is a method comprising creating an electrical charge differential between a first point and a second point separated from the first point by an electrically conductive medium comprising a biological cell. A first electrical parameter between the first and second points is then measured. A second electrical parameter is then adjusted based on the measuring of the first electrical parameter. The first electrical parameter may be any parameter such as one selected of the group consisting of current, voltage and electrical impedance. The second electrical parameter may be any parameter (the same as or different from the first electrical parameter) such as one selected from the group consisting of current, voltage or a combination of current and voltage.
In a preferred embodiment the method further includes placing a material in the electrically conductive medium, and adjusting the second electrical parameter in order to move the material into the biological cell. The material placed within the electrically conducted medium may be any material such as a pharmaceutically active compound or drug, a nucleotide sequence, a fluorescent dye, or a crystal which is specifically designed to effect the cell in a desired manner. In accordance with the method various conditions are adjusted so that the electrical potential between the two points is sufficiently high so as to cause the cell to be permeabilized. However, the conditions between the two points are further adjusted so that electroporation is reversible and as such does not cause cell death unless that is a result specifically being sought.
In another aspect of the invention the electroporation is not carried out for the purpose of moving material into or out of a cell but rather to analyze the cell or group of cells and provide information or diagnosis of the tissue or individual which contains the tissue. In accordance with this method an electrical charge differential is created between a first point and a second point separated from the first point by an electrically conducted medium comprising a biological cell. A first electrical parameter is then measured between the first and second points. The measuring of the first electrical parameter is then analyzed in order to determine a character of the cell and in particular a characteristic of a membrane of the cell. The first electrical parameter may be any parameter and is preferably selected from the group consisting of current, voltage and electrical impedance. A second electrical parameter is preferably adjusted in a manner which effects the membrane of the cell or cells present in the medium and the second electrical parameter is any parameter but preferably selected from current, voltage or a combination of both.
Another aspect of the invention is the device which is preferably comprised of a first electrode, a second electrode, a source of electricity which may later be connected to the electrodes but is optionally present when the device is sold. The device further includes a means for hindering the flow of electrical current between the first and second electrodes except for electrical current flow through a defined route. Further, the device includes a means for measuring an electrical parameter such as current, voltage or electrical impedance through the defined route and a means for adjusting the source of electricity based on the measured electrical parameter. The means for hindering electrical current flow is preferably comprised of a non-conductive material and defined route comprised of one or more openings each with a diameter less than that of a biological cell so that a cell can fit within the defined route and have a current flow through but preferably not around the cell.
The device and systems of the invention can be used within the method in order to move a wide range of materials into or out of the biological cell in order to obtain a desired result. The process can be carried out on an individual cell, a group of cells, cells within a cell culture or within a living organism, e.g. cells within invertebrates and vertebrates including mammals as well as in plants. When carrying out the process on a plurality of cells (e.g. a tissue) a process of imaging the tissue and adjusting electrical current in real time based on images may be used. An imaging technology which may be applied is electrical impedance tomography (EIT). This technology relies on differences in bioelectrical attributes within the body or an organism (e.g. a human) to produce an image. In the method of the invention EIT images can be used in the same manner as the measuring step is used when the process is carried out on a single biological cell. In essence, the EIT technology makes it possible to xe2x80x9cseexe2x80x9d the effect of increased electrical current flow resulting from electroporation thereby providing information which can be used to precisely adjust the flow of electrical current so that cell membranes are permeabilized while not permanently disrupted.
Another aspect of the invention is a method which comprises sending an electrical current between a first point and a second point separated by the first point by an electrically conductive medium comprising tissue. The tissue may be present within a living organism such as a vertebrate or invertebrate and specifically includes mammals and humans. After the current is sent an image of the tissue is created wherein the image is based on an electrical parameter such as the electrical impedance of the tissue. Using the image as a guide an electrical parameter is adjusted in order to obtain a desired degree of electroporation of biological cells in the tissue. Electroporation will change electrical impedance and that change can be visualized on the image created. The electrical parameter adjusted may be any parameter such as current, voltage or a combination of both. In a preferred embodiment a material is placed in the electrically conducted medium such as being injected into the tissue and the adjustment of the current is carried out, based on the image, in a manner so as to move the material into biological cells of the tissue. The image created is preferably an impedance image created from known current inputs and measured input voltage using a reconstruction algorithm. The impedance image may be created from a known voltage input, a measured current input, or combination of known voltage input and measured current input.
A device for carrying out this method is another aspect of the invention which device includes a means for creating an electrical current across an electrically conducted medium. The device further includes a means for analyzing a first electrical parameter of the electrically conductive medium in order to create an image and a means for adjusting a second electrical parameter based on the image to obtain a desired degree of electroporation of biological cells in the electrically conductive medium. The first electrical parameter is preferably electrical impedance and the second electrical parameter is preferably selected from the group consisting of current, voltage or a combination of both. The current is preferably created by a plurality of electrodes positioned about an area of tissue upon which the electroporation is to be carried out.
The present invention arises in part from the discovery that the onset and extent of electroporation in a biological cell can be correlated to changes in the electrical impedance (which term is used herein to mean the ratio of current to voltage) of the biological cell or of a conductive medium that includes the biological cell. An increase in the current-to-voltage ratio across a biological cell occurs when the cell membrane becomes permeable due to pore formation or because of cell damage or other modes of cell membrane poration. Likewise, a decrease in the current-to-voltage ratio through a flowing conductive fluid occurs when the fluid draws a biological cell into the region between the electrodes in a flow-through electric cell. Thus, by monitoring the impedance of the biological cell or of an electrolyte solution in which the cell is suspended, one can detect the point in time in which pore formation in the cell membrane occurs, as well as the relative degree of cell membrane permeability due to the pore formation. This information can then be used to establish that a given cell has in fact undergone electroporation, or to control the electroporation process by governing the selection of the electrical parameters of the process e.g. the voltage magnitude. This discovery is also useful in the simultaneous electroporation of multitudes of cells in a cell culture or in vertebrates, invertebrates or plants. Specific embodiments apply the invention to mammals including humans. The process provides a direct indication of the actual occurrence of electroporation and an indication of the degree of electroporation averaged over all the cells being subjected to the process. The discovery is likewise useful in the electroporation of biological tissue (masses of biological cells with contiguous membranes) for the same reasons.
The benefits of this process include a high level of control over the onset and degree of electroporation, together with a more detailed knowledge of the occurrence and degree of permeability created in particular individual cells or cell masses. When applied to individual cells or to a succession of individual cells, this process assures that the individual cells are indeed rendered permeable and are indeed transformed by the introduction of chemical species. The process also offers the ability to increase the efficiency of electroporation by avoiding variations in the electrical environment that would destroy some cells while having an insufficient effect on others.
The invention can be understood by describing a simple embodiment which involves the use of an electrical device or system in which a biological cell can be placed and that contains a barrier that directs the electric current flow and hence the ion flow through a flow path that passes through the biological cell while permitting substantially no electric current to bypass the biological cell. In some of these embodiments, the invention involves the use of an apparatus containing two liquid-retaining chambers separated by a barrier that is substantially impermeable to an electric current. The barrier contains an opening that is smaller than the biological cell such that the biological cell once lodged in the opening will plug or close the opening. To achieve electroporation, the biological cell is secured over the opening by mechanical, chemical and/or biochemical means, preferably in a reversible manner so that the biological cell can later be removed without damage to the biological cell. Once the biological cell is secured over the opening, a voltage is imposed between the two chambers and across the biological cell residing in the opening. The passage of current between the chambers is thus restricted to a path passing through the opening and hence through the biological cell. By monitoring the current-voltage relation in the electric cell, the onset of electroporation is detected and the degree of pore formation is controlled, to both assure that electroporation is occurring and to prevent excessive pore formation and cell death. The user is thus afforded a highly precise knowledge and control of the condition of and the flux across the biological cell membrane.
In another series of embodiments, this invention is useful in the diffusive transport of chemical species into or out of a biological cell. In these embodiments, the cell is again divided into two chambers separated by a barrier, and the biological cell is lodged across an opening in the barrier in such a manner that the passage of liquid around the cell from one chamber to the other is substantially prevented. A liquid solution of the species to be introduced into the biological cell is placed in one or both of the chambers. The concentration of the species in the solution differs from that in the cell (either higher or lower, depending on whether one seeks to introduce or remove the species from the cell), or the concentration in one chamber differs from that in the other chamber.
In preferred methods of applying this invention to diffusive transport, the solutions in the two chambers differ in concentration such that the driving force for the diffusive transport is between the two chambers themselves rather than between the chambers and the interior of the biological cell. Knowledge and controlled monitoring of the concentrations in each of the two chambers on a periodic or continuous basis as the diffusion proceeds, together with the precise knowledge of the dimensions of the opening, enables the user to precisely observe and control the rate and amount of the species that enters the cell. The diffusion time can be controlled by imposing stepwise changes in the concentrations in either or both of the chambers, thereby imposing or removing the concentration differential. An application of particular interest is the combination of this type of diffusive transport of a chemical species with controlled electroporation as described in the preceding paragraph.
In addition to being useful in connection with electroporation technology the present invention can provide valuable information relating to a cell or group of cells or tissue containing a group of cells by monitoring electrical impedance and thereby providing information regarding the integrity of a cell membrane. Specifically, measurements are carried out regarding the movement of charged particles across a cell membrane. These measurements are related to the amount of electrical current needed to carry out the diffusion across a cell membrane. The information obtained can be analyzed directly or compared to previous measurements of a same tissue or measurements carried out on diseased or normal tissue thereby providing an indication of the amount of change which has occurred in the tissue being measured (based on an earlier measurement of the same tissues) or the amount of variance between the tissue being measured and tissue with impaired cell membranes (e.g. diseased cells) or a normal cell or tissue. The method is carried out in a manner similar to that used for conducting electroporation. However, no material needs to be added to the medium surrounding the cells. The device is similar in that it is divided into two portions with a positive electrode on one side and a negative electrode on another side separated by a barrier with the cells being positioned along openings on the barrier in a manner which allows for the passage of charged particles through the cell and through the opening in the barrier from one electrode to another. The barrier hinders or completely eliminates the flow of charged particles except through the openings. The measurement of electrical impedance between the electrodes make it possible to distinguish between cells with an intact membrane and cells with impaired membranes. By more precisely carrying out the measurements it is possible to make determinations with respect to the integrity of a normal cell membrane relative to an impaired (e.g. diseased) cell membrane.
Each of the various embodiments of this invention may be used with two or more (i.e. a plurality of) biological cells simultaneously, or cell masses such as in tissue which may be in an animal or plant during the process. The apparatus described above can be adapted for use with two or more biological cells by arranging the barrier such that the current or diffusive transport will be restricted to a flow path that passes through all of the cells while preventing bypass around the cells. A further application of the concepts of this invention is the electroporation of biological cells suspended in a flowing liquid. Electrodes are placed in fixed positions in the flow channel, and a voltage is imposed between the electrodes while current passing between the electrodes is monitored. Biological cells entering the region between the electrodes will lower the current, the impedance serving as an indication of the presence of one or more cells in the region, and optionally also as a signal to initiate the application of a higher voltage sufficient to achieve electroporation.
A further application of the device, system and method of the invention is the electroporation of biological cells present within a tissue which tissue may be present within a living organism such as a mammal. Electrodes are placed in fixed positions within the tissue, and voltage is applied between the electrodes while current passing between the electrodes is monitored. Biological cells with intact membranes in the region between the electrodes will increase the electrical impedance. Accordingly, a measurement of the electrical impedance provides an indication of the presence of one or more cells in the region. Electroporation will decrease the measured amount of impedance. When the process is carried out on a tissue then the measurement of electrical impedance is a statistical average of the cells present between the electrodes.
Electroporation methodology of the invention can be carried out on tissue in a living organism using an imaging technology which makes it possible to determine when (and preferably to some extent the degree) cell membranes are transformed so as to allow the flow of electrical current through their membranes. The preferred imaging technology is electrical impedance tomography (EIT) which provides a changing image created from information on differences in bio-electrical attributes of the tissue being imaged. A typical EIT image is acquired by injecting electrical currents into the body and measuring the resulting voltages through an electrode array. An impedance image is then produced from the known current inputs and the measured voltage data using a reconstruction algorithm. EIT is particularly appropriate for the implementation of the invention in tissue because it actually maps electrical impedances. Therefore, the region of tissue that will undergo electroporation and in which, consequently, the equivalent electrical impedance of the cells will change will be imaged by EIT. The image is used to adjust the electrical parameters (e.g. flow of electrical current) in a manner which allows electroporation to occur without damaging cell membranes.
Among the advantages that this invention offers relative to the prior art are the ability to treat cells individually and to adapt the treatment conditions to the needs of individual cells. In embodiments where voltage is applied, the monitoring of the impedance affords the user knowledge of the presence or absence of pores and shows the progress of the pore formation and whether irreversible pore formation that might lead to cell death has occurred.
An advantage of the barrier-and-opening apparatus is the high sensitivity of the signal to noise ratio by virtue of its restriction of the current to a current flow path passing through the opening.
A still further advantage is the ability of the apparatus and method to be integrated into an automated system whereby the condition of each cell is monitored by instrumentation and individual cells are lodged in the opening and then removed at times governed by the monitored conditions.
An aspect of the invention is a method of controlling electroporation of biological cells in real time by adjusting an electrical parameter (e.g. voltage and/or current) applied to a system based on real time measurements of changes in current detected.
A feature of the invention is that the general concepts can be applied to carry out electroporation on a cell, multiple cells, a tissue or areas of tissues in a living animal.
An advantage of the invention is that a precise amount of electroporation can be obtained and cell damage avoided by controlling any given electrical parameter (e.g. current and/or voltage) applied based on real time measurements of changes in current which relates to the amount of electroporation being obtained.
Another advantage of the invention is that it can be used to transfect cells with nucleotide sequences without the need for packaging the sequences in a viral vector for delivery, thereby avoiding the cellular specificities of such vectors.
Still other advantages are that the process can be carried out relatively quickly with a relatively low degree of technical expertise.
Yet another advantage is that the process can be used to transfect cells without generating an immune response.
Still another advantage is that the process is not limited by the size of the DNA (i.e. the length of the DNA sequences) and the amount of DNA brought into a cell can be controlled.
Another feature of the invention is that imaging technologies such as EIT can be used to detect changes in impedance in a volume of cells.
Another feature of the invention is that it can use EIT in order to map impedance of an area of tissue and thereby detect changes in cell impedance in a volume of cells to adjust any given electrical parameter (e.g. current flow and/or voltage) to obtain desired electroporation.
These and further features, advantages and objects of the invention will be better understood from the description that follows.